Elevation of plasma homocyst(e)ine (H(e)) is associated with increased risk for atherosclerotic vascular disease. We have observed impaired vascular function in monkeys with moderate hyperhomo-cyst(e)inemia (plasma H(e) > 10 fM) (J Clin Invest 98:24-29, 1996). In this study we tested the hypothesis that hyperhomocyst(e)inemia contributes to vascular dysfunction in atherosclerotic monkeys. Cynomolgus monkeys were fed an atherogenic diet that is known to produce both hypercholesterolemia and moderate hyperhomocyst(e)inemia. After 16 months, the atherogenic diet was supplemented with B vitamins daily (5 mg folic acid, 400 fg B12 and 20 mg B6) for 6 months. Plasma H(e) decreased from 12.8 q 2.8 to 3.5 q 0.3 fM (n=9; mean q SE); p <0.01) after addition of vitamins, but plasma cholesterol did not change (522 q 63 vs. 514 q 41 mg/dL; p >0.05). In response to intraarterial collagen, blood flow to the leg decreased by 30 q 3% and 38 q 5%, respectively, before and after vitamin supplementation (p <0.05). In vivo responses to endothelium-dependent vasodilators (acetylcholine or ADP) were impaired at baseline and did not improve after addition of vitamins. In the carotid artery ex vivo, relaxation to acetylcholine improved only slightly after vitamin supplementation. Ex vivo thrombomodulin anticoagulant activity was 3-fold higher in monkeys fed the atherogenic diet (with or without B vitamins) than in normal monkeys (p <0.05). We conclude that atherosclerosis in monkeys is associated with elevated thrombomodulin activity, and that normalization of plasma H(e) is not sufficient to restore normal vascular function during persistent hypercholesterolemia.